1. Field of the Invention
The present invention relates to methods for the identification and use of chemical compounds that can selectively kill tumor cells by inducing cell apoptosis. Apoptosis is a natural process for normal cells and means programmed cell death. Tumor cells can avoid apoptosis and thus are able to survive and develop in the body. The method is significant in cancer therapy, since it can detect novel candidate chemotherapeutic drugs that could be used for the clinical treatment of cancer patients. In particular, the present invention relates to the methods using chemical compounds that are targeted to inhibit 12-lipoxygenase in inducing the tumor cell apoptosis.
2. Description of Related Art
Normal tissue homeostasis is maintained by balanced cell proliferation and cell death, which occurs most frequently in the form of apoptosis or programmed cell death. Tumor cells differ significantly from their normal counterparts with respect to the control of cell growth and proliferation. Most tumor cells demonstrate a self-dominant growth pattern either due to their abnormal response to environmental stimuli (hormones, growth factors, cytokines, etc.) or due to an autonomous nature of growth (i.e., autocrine stimulation). Tumor cells also demonstrate abnormal apoptotic responses. Many factors have been shown to regulate apoptosis, including (i) growth factors and growth factor receptors such as retinoid acid, interleukin-3, stem cell factor, interferon-.gamma., erythropoietin, NGF/NGF (nerve growth factor) receptor, TNF-.alpha./Fas (tumor necrosis factor-.alpha.), steel factor/Kit receptor, TGF-.beta./TGF (transforming growth factor) receptor, insulin, EGF/EGFR (epidermal growth factor), IGF-1/IGF (insulin-like growth factor) receptor, and PDGF/PDGF (platelet-derived growth factor receptor); (ii) intracellular signal transducers such as protein kinase C, PI-3 (phosphoinositol-3) kinase, Ras and GTPase, PLC-.gamma. (phospholipase C-.gamma.), tyrosine kinases and protein phosphatases, lipid signaling molecules such as eicosanoids, sphingosine, ceramide, and Ca.sup.2+ ; (iii) cell cycle regulators exemplified by Cdc-2 and E2F-1; (iv) reactive oxygen species or other free radicals; (v) extracellular matrix regulators/cell adhesion molecules (extracellular matrix proteins such as fibronectin and transmembrane integrin receptors) ; and (vi) specific endonucleases such as Ca.sup.2+ - and Mg.sup.2+ -dependent DNase and cytoplasmic proteases typified by ICE (interleukin 1.beta.-converting enzyme) family. Many of these regulators have been associated with various human malignancies and apoptosis. For example, studies on human tumors including neuroblastoma, glioma, lymphoma, breast carcinoma, colorectal adenocarcinoma, melanoma and gastrointestinal malignancies have demonstrated an overall positive correlation between increased expression of Bcl-2 (or Bcl-X.sub.L) or decreased expression of Bax and uncontrolled tumor cell growth, and, in some cases, with tumor progression and a poor prognosis of cancer patients. Another example is p53, a phosphoprotein known to modulate gene transcription, police cell cycle checkpoints, control DNA replication and repair, and maintain genomic stability. Wild type p53 also positively regulates apoptosis. p53 gene mutations have been linked to attenuated apoptosis in multiple cancers represented by Wilms' tumor, colon cancer, cervical carcinoma and breast cancer. Since apoptosis plays a critical role in multiple steps (transformation, progression and survival of metastases) of tumorigenesis as well as in tumor cells' response to chemotherapeutic drugs or radiation therapy, many chemoprevention and therapeutic regimens attempting to manipulate apoptotic process have been proposed to aid in the clinical treatment of cancer patients (Fesus, L., et al., J. Cell Biochem. 22:151-161 (1995); Lotan, R., J. Natl. Cancer Inst. 87:1655-1657 (1995); van Zandwijk, N., J. Cell Biochem. 22:24-32 (1995)).
Arachidonic acid (AA) is an essential component of the cell membrane phospholipids. AA released through the action of phospholipase A.sub.2 is metabolized via three major biochemical pathways: (i) the cyclooxygenase (COX) pathway leading to the generation of prostaglandins, prostacyclin, and thromboxane; (ii) the lipoxygenase (LOX) pathway giving rise to various hydroperoxy (HPETEs) and hydroxy (HETEs) fatty acids as well as leukotrienes; and (iii) the P450-dependent epoxygenase pathway generating EETs. Mammalian LOX display varying degrees of substrate specificity for insertion of molecular oxygen into arachidonic acid at carbon positions 5, 12, and 15. The enzymes, based on the abundance of the majority products have thus been termed 5, 12, and 15 lipoxygenases, respectively. The 12-LOX catalyzes the transformation of AA into 12(S) -hydroperoxyeicosatetraenoic acid (12-HPETE) and its 12(S)-hydroxy derivatives i.e. 12(S)-HETE!. Three types of mammalian 12-LOX enzymes have so far been reported. The first is human platelet-type 12-lipoxygenase expressed normally in platelets, HEL (human erythroleukemia) cells, and umbilical vein endothelial cells (Funk, C. D., et al., Proc. Natl. Acad. Sci. 87:5638-5642 (1990); Funk, C. D., et al., Proc. Natl. Acad. Sci. 89:3962-3966 (1992)). Platelet-type 12-LOX metabolizes only AA (but not C-18 fatty acids such as linoleic acid) to form exclusively 12(S)-HETE (Funk, C. D., et al., Proc. Natl. Acad. Sci. 87:5638-5642 (1990) Marnett, L. J., et al., Adv. Prostaglandin Thromboxane Leukotriene Res. 21:895-900 (1990)). The second is porcine leukocyte-type 12-LOX which metabolizes both AA and linoleic acid thus generating 12(S)-HETE as well as small amounts of 15(S)-HETE (Hada, T., et al., Biochim. Biophys. Acta 1083-1087 (1991)). The third type of 12-LOX (sometimes termed epithelial 12-lipoxygenase) has been isolated from bovine tracheal epithelial cells (De Marzo, N., et al., J. Physiol. 262:L198-L207 (1992)); rat brain (Watanabe, S., et al., Eur. J. Biochem. 212:605-612 (1993)), and murine macrophages (Freier-Moar, J., et al., Biochim. Biophys. Acta., 1254:112-116 (1995)), which shares more homology with 15-LOX and leukocyte-type 12-LOX than with platelet-type 12-LOX. This type of 12-LOX, like reticulocyte 15-LOX and leukocyte-type 12-LOX, catalyzes the formation of both 12(S)-HETE and 15(S)-HETE.
The role of AA metabolites in regulating cell proliferation has been recognized for more than two decades. Numerous studies have demonstrated a strong positive correlation between growth factor- (EGF, insulin, PDGF, etc.) promoted cell proliferation and generation of various COX products, primarily prostaglandins (Skouteris, G. G., et al., Biochem. Biophys. Res. Commun., 178:1240-1246 (1991); Nolan, R. D., et al., Mol. Pharmacol. 33:650-656 (1988); Smith, D. L., et al., Prostaglandins Leukotrienes Med., 16:1-10 (1984)). Similarly, it has been found that various eicosanoids derived from LOX pathways as well as epoxygenase pathways of AA metabolism play an essential role in mediating the growth factor-stimulated normal cell and tumor cell growth. Examples include 15-HETE as a mitogenic regulator of T-lymphocyte (Bailey, J. M., et al., Cell Immunol., 67:112-120 (1982)), 12-HETE and LTB.sub.4 as growth stimulators of epidermal cells (Chan, C., et al., J. Invest. Dermatol., 85:333-334 (1985)), 12-HETE stimulation of keratinocyte DNA synthesis (Kragballe, K., et al., Arch. Dermatol. Res., 278:449-453 (1986)), 15-/12-HETEs as mediators of insulin and EGF-stimulated mammary epithelial cell proliferation (Bandyopadhyay, G. K., et al., J. Biol. Chem., 263:7567-7573 (1988)) and as synergistic effectors of bFGF- (basic fibroblast growth factor) and PDGF-regulated growth of vascular endothelial cells and smooth muscle cells (Yamaja Setty, B. N., et al., J. Biol. Chem., 262:17613-17622 (1987); Dethlefsen, S. M., et al., Exp. Cell Res., 212:262-273 (1994)), 12(S)-HETE as a regulator of EGF- and insulin-stimulated DNA synthesis and protooncogene expression in lens epithelial cells (Lysz, T. W., et al., Cell Growth & Differ., 5:1069-1076 (1994)) and as the mediator of angiotensin II-induced aldosterone synthesis in adrenal glomerulosa cells (Nadler, R. D., et al., J. Clin. Invest., 80:1763-1769 (1987)).
The present inventors have long been interested in the modulatory role of various eicosanoids in tumorigenesis and metastatic process. Early work demonstrated an important function for prostacyclin (PGI.sub.2) and thromboxane (TxA.sub.2), two major cyclooxygenase (COX) products of AA metabolism derived primarily from vascular endothelial cells and platelets, respectively, in regulating the hematogenous spreading of malignant tumor cells (Honn, K. V., et al., Science 212:1270-1272 (1981); reviewed in Schneider et al., Cancer metastasis Rev. 13:349-364 (1994)). Later, systematic in vitro and in vivo studies have led to the discovery that many LOX metabolites also play a key role in modulating the phenotypic properties of tumor cells as well as tumor cell-vasculature interactions (Reviewed in Honn, K. V., et al., Cancer Metastasis Rev. 11:353-375 (1992); Honn, K. V., and D. G. Tang, Seminar Thromb. Hemost., 18:392-415 (1992); Tang, D. G. and K. V. Honn, Invasion Metastasis 14:109-122 (1995)). Prominently, a small hydroxy fatty acid molecule derived from the LOX pathway of AA metabolism, i.e., 12(S)-HETE 12(S)-hydroxyeicosatetraenoic acid!, has been observed to possess a wide-spectrum of biological activities including, among others, inducing platelet aggregation, stimulating insulin secretion, suppressing renin production, chemoattracting leukocytes, facilitating macrophage adhesion, inhibiting prostacyclin biosynthesis by vascular endothelial cells (Spector, A. A., et al., Prog. Lipid Res. 27:271-323 (1988); Sekiya, K., et al., Biochem. Biophys. Res. Commun. 105:1090-1095 (1982), modulating tumor cell interactions with extracellular matrix, promoting tumor cell motility, facilitating tumor cell release of proteolytic enzyme cathepsin B, reorganizing tumor cell cytoskeleton, promoting tumor cell adhesion on endothelial cells via upregulating integrin expression on tumor cells and/or endothelial cells, and inducing endothelial cell retraction thus enhancing tumor cell extravasation from the vasculature (reviewed in Honn, et al., Seminar Thromb. Hemost. 18:390-413 (1992); Honn, K. V., et al., Cancer Metastasis Rev., 13:365-396 (1994); Tang, D. G. and K. V. Honn, Annals New York Acad. Sci., 744:199-215 (1994); Tang, D. G. and K. V. Honn, Invasion Metastasis 14:109-122 (1995)). Very recently, the inventors have reported 12(S)-HETE as a mitogenic factor for microvascular endothelial cells (Tang, D. G., et al., Biochem. Biophys. Res. Commun. 211:462-468 (1995a)) and a transactivator of integrin .alpha.v (Tang, D. G., et al., J. Cell Sci. 108:2629-2644 (1995b)). Significant progress has been made in delineating the molecular mechanisms of the 12(S)-HETE effects. 12(S)-HETE, possibly through binding to a cell surface receptor(s), triggers phosphoinositol lipid hydrolysis (Liu, B., et al., Proc. Natl. Acad. Sci. 92:9323-9327 (1995)) leading to the intracellular activation of protein kinase C (PKC; Liu, B., et al., Cell Regul. 2:1045-1055 (1992)) and/or protein tyrosine kinase (PTK; Tang, D. G., et al., J. Cell. Physiol. 165:291-306 (1995c)). The interactions of these phosphorylated protein kinases with various intracellular molecular targets (e.g., cytoskeletal proteins, adhesion molecules, signaling molecules, etc.) largely explain the versatility of the 12(S)-HETE effects.
Both COX and LOX products of AA metabolism may also be involved in modulating tumor cell survival and apoptosis. Thus, many prostaglandins such as PGE.sub.2 (prostaglandin E.sub.2) (Brown, D. M., et al., Clin. Immunol. Immunopathol. 63:221-229 (1992) ), PGA.sub.2 and .DELTA..sup.12 -PGJ.sub.2 (Kim, I-K, et al., FEBS Lett. 312:209-214 (1993)) have been shown to induce apoptosis of leukemia or lymphoma cells as well as solid tumor cells. Similarly, TxA.sub.2 induces apoptotic cell death of immature thymocytes by binding to the cell surface TxA.sub.2 receptors (Ushikubi, F., et al., J. Exp. Med. 178:1825-1830 (1993) ). Interestingly, PGE.sub.2 also has been reported to protect cells from apoptosis induction (Goetzel, E. J., et al., J. Immunol. 154:1041-1047 (1995)). On the other hand, various COX inhibitors (NSAID) (Non-steroidal anti-inflammation drugs) have been consistently demonstrated to trigger apoptosis of cultured cells. Thus, indomethacin, sulindac sulfide and sulfone inhibit colon carcinoma cell (HT-29) growth by inducing apoptosis (Shiff, S. J., et al., J. Clin. Invest. 96:491-503 (1995); Piazza, G. A., et al., Cancer Res. 55:3110-3116 (1995); Shiff, S. J., et al., Exp. Cell Res. 222:179-188 (1996)). Likewise, multiple NSAIDs including diflunisal, indomethacin, acemethacin, diclofenac, mefenamic acid, flufenamic acid, niflumic acid, ibuprofen, and carprofen cause apoptosis in chicken embryo fibroblasts (Lu, X., et al., Proc. Natl. Acad. Sci. 92:7961-7965 (1995)). These observations suggest that the COX/COX metabolites may play a dual role in regulating cell survival and apoptosis. Under certain circumstances the COX products (e.g., PGE.sub.2 and cyclopentenone prostaglandins) can either directly trigger cell death or mediate apoptosis induced by, e.g., TNF-.alpha. (Larrick, J. W., and S. C. Wright, FASEB J. 4:3215-3223 (1990)). In a different scenario, the COX activity/function are critical for cell survival since inhibition with various inhibitors leads to cell death (see above). Consistent with this, overexpression of COX-2 has been observed to confer resistance in rat intestinal epithelial cells to apoptosis induction by butyrate (Tsujii, M., and R. DuBois, Cell 83:493-501 (1995)).
Similar to the COX system, some LOXs and their products may also play a dual regulatory role in cell survival and apoptosis. Exogenous lipid hydroperoxides such as 15-HPETE induces HIV-infected human T cells (Sandstrom, et al., J. Biol. Chem. 269:798-802 (1994)) due to their inability to convert 15-HPETE to 15-HETE owing to a reduction in the glutathione peroxidase activity, LOX metabolites have been proposed as the actual mediators of TNF.alpha.-induced apoptosis of multiple cells since LOX inhibitors such as ETYA and NDGA could inhibit its cytotoxicity (Chang, D. J., et al., Biochem. Biophys. Res. Commun. 188:538-546 (1992); O'Donell, V. B., et al., Biochem. J. 310:133-141 (1995)). On the other hand, 5-LOX inhibitors can cause apoptosis of human leukemia blast cells (Anderson, K. M., et al., Prosta. Leuko. Essent. Fat. Acids 48:323-326 (1993)) and interruption of 5-LOX-mediated growth factor signaling inhibits the growth of lung cancer cells due to apoptosis induction (Avis, I. M., et al., J. Clin. Invest. 97:806-813 (1996)), suggesting that in some cells 5-LOX pathway may function as a critical survival factor.
There has been no literature report relating to the potential role of mammalian 12-LOX in regulating tumor cell survival and apoptosis. Based on extensive studies by the inventors on the role of 12(S)-HETE in regulating tumor cell growth and on the relationship between 12-LOX expression and the metastatic capacity of a variety of tumor cells, it is hypothesized that 12-LOX system may also be involved in regulating tumor cell survival and apoptosis.